The present invention relates to a chemically modified polypeptide in which at least one of the hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative; a method for producing the modified polypeptide; a method of treating a patient having reduced granulocytes or thrombocytes using the modified polypeptide; a composition for the treatment comprising the modified polypeptide; and use of the modified polypeptide.
A chemically modified polypeptide in which at least one of amino groups, carboxyl groups, mercapto groups or guanidino groups in the polypeptide molecule is modified with a polyalkylene glycol derivative (WO 95/023165) and modification of free thiol groups of cysteine residues in the polypeptide molecule are known (EP 0668353). However, when at least one of such amino groups, carboxyl groups, mercapto groups, guanidino groups or free thiol groups of cysteine residues in the polypeptide molecule is modified with a polyalkylene glycol derivative, the activity of the polypeptide may be markedly or completely lost.
For example, the activity of interleukin-15 completely disappears when its amino group(s) are modified with polyethylene glycol [J. Biol. Chem., 272:2312 (1997)].
Nothing is known about a chemically modified polypeptide in which at least one of the hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative.
Great attention has been directed toward the development of
(1) a method for the analysis of influences of hydroxyl groups upon the activity of a polypeptide, in a case where the hydroxyl group concerned is in the active site of the polypeptide,
(2) a novel chemical modification method which can avoid a probable case where the biological activity of a polypeptide is considerably spoiled when the polypeptide is treated by a conventional chemical modification method, and a chemically modified polypeptide obtained by the method, and
(3) a novel method which can improve resistance of polypeptide against protease, freezing-thawing and denaturing agents.
The present invention relates to a chemically modified polypeptide in which at least one of the hydroxyl groups in the polypeptide molecule is modified with a polyalkylene glycol derivative; a method for producing the modifying polypeptide; a method of treating a patient having reduced granulocytes or thrombocytes using the modifying polypeptide; use of the modifying polypeptide; a pharmaceutical preparation comprising the modifying polypeptide; and a composition for the treatment comprising the modifying polypeptide.
With regard to the polypeptide which can be used in the present invention, any polypeptide can be used so long as it contains a hydroxyl group and has a physiological activity or a pharmacological activity. Examples include those having an activity, such as asparaginase, glutaminase, uricase, superoxide dismutase, lactoferin, streptokinase, plasmin, adenosine deaminase, interleukin-1 to 13, interleukin-15, interferon-xcex1, interferon-xcex2, interferon-xcex3, human granulocyte colony-stimulating factor (hereinafter referred to as xe2x80x9chG-CSFxe2x80x9d), and the like.
Examples of a polypeptide having an hG-CSF activity include a polypeptide comprising the amino acid sequence represented by SEQ ID NO:1, a polypeptide comprising a partial amino acid sequence of the sequence, a polypeptide comprising an amino acid sequence in which some parts of amino acids of the sequence are substituted by different amino acids [Nature, 319:415 (1986), Japanese Published Unexamined Patent Application No. 267292/88, Japanese Published Unexamined Patent Application No. 299/88, WO 87/01132] and the like. Specific examples of the polypeptide comprising an amino acid sequence in which some parts of amino acids of the amino acid sequence represented by SEQ ID NO:1 are substituted by different amino acids (hG-CSF derivatives) are shown in Table 1.
Examples of the polyalkylene glycol derivative include polyethylene glycol derivatives, polypropylene glycol derivatives, polyethylene glycol-polypropylene glycol copolymer derivatives, and the like.
The chemically modified polypeptide of the present invention can be produced using a chemical modifying agent comprising the above polyalkylene glycol derivative, and compounds represented by the following formula (I) can be exemplified as preferred chemical modifying agents.
The compounds include polyalkylene glycol derivatives represented by
R1xe2x80x94(M)nxe2x80x94Xxe2x80x94R2xe2x80x83xe2x80x83(I) 
{wherein R1 represents an alkyl group or an alkanoyl group; M represents
xe2x80x94OCH2CH2xe2x80x94, xe2x80x94OCH2CH2CH2xe2x80x94
or
xe2x80x94(OCH2CH2)rxe2x80x94(OCH2CH2CH2)sxe2x80x94
(wherein r and s are the same or different, and each represents an optionally changeable positive integer); n is an optionally changeable positive integer; X represents a bond, O, NH or S; and R2 represents 
 less than wherein R3 represents OH, halogen or
xe2x80x94Xaxe2x80x94(Ma)naxe2x80x94R1a 
(wherein Xa, Ma, R1a and na each has the same meanings as the above X, M, R1 and n, respectively); and Y represents halogen or
xe2x80x94Zxe2x80x94(CH2)pxe2x80x94(O)mxe2x80x94W 
[wherein Z represents O, S or NH; and W represents a carboxyl group or a reactive derivative thereof, or 
(wherein R4 represents an alkyl group, and Hal represents halogen); p is an integer of 0 to 6; and m is 0 or 1] greater than ,
xe2x80x94(CO)mxe2x80x94(CH2)txe2x80x94W 
(wherein t is an integer of 0 to 6; and m and W have the same meanings as defined above), 
(wherein Hala, pa and R4a each has the same meanings as the above Hal, p and R4, respectively), 
(wherein R3 and W have the same meanings as defined above), 
(wherein R3, t and W have the same meanings as defined above), 
(wherein W has the same meaning as defined above), 
(wherein R5 represents a residue in which an amino group and a carboxyl group are removed from an amino acid; and W has the same meaning as defined above)}.
With regard to the above compound represented by formula (I), examples of the alkyl group represented by R1, R4 or the like include straight or branched alkyl groups having 1 to 18 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, isooctyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like; examples of the alkanoyl group represented by R1 include straight or branched alkanoyl groups having 1 to 18 carbon atoms, such as formyl, acetyl, propionyl, butyryl, valeryl, pivaloyl, pentanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, and the like; examples of the halogen represented by R3, Y, Hal or the like include chlorine, bromine and iodine atoms; examples of the reactive derivative of a carboxyl group represented by W or the like include acid halides, such as an acid chloride, an acid bromide, and the like, active esters, such as a p-nitrophenyl ester, an N-oxysuccinimide ester, and the like, and mixed acid anhydrides with monoethyl carbonate, monoisobutyl carbonate and the like; and examples of the amino acid represented by R5 include glycine, L-alanine, L-valine, L-leucine, L-serine, D-alanine, D-valine, D-leucine, D-serine, xcex2-alanine, and the like. The positive integer represented by n, r or s is 1 to 20,000, preferably 50 to 5,000 for n, and 1 to 5,000 for r and s.
The polyalkylene glycol derivatives have a molecular weight of 500 to 1,000,000, preferably 3,000 to 1,000,000.
A plurality of hydroxyl groups may be present in the polypeptide molecule, and chemical modification of at least one of these groups may be sufficient when the polypeptide is chemically modified.
Examples of the hydroxyl group in the polypeptide molecule include a hydroxyl group of a serine or threonine residue, preferably a hydroxyl group of a serine residue.
The polypeptide can be chemically modified by reacting a chemical modifying agent, such as a polyalkylene glycol derivative selected from a group consisting of polyethylene glycol derivatives, polypropylene glycol derivatives, polyethylene glycol-polypropylene glycol copolymer derivatives and the like, with a polypeptide having a hydroxyl group.
Examples of the method for reacting the hydroxyl group in a polypeptide molecule with a chemical modifying agent such as polyethylene glycol derivatives or polypropylene glycol derivatives include the methods described in Japanese Published Unexamined Patent Application No. 316400/89, Biotech. Lett., 14:559-564 (1992), BIO/TECHNOLOGY, 8:343-346 (1990), and the like, and modified methods thereof. That is, specific examples of the methods which can be used include methods wherein a polyethylene glycol derivative or a polypropylene glycol derivative is added to an aqueous solution of a protein which has been adjusted to a pH of 6 to 10 in an amount of 1 to 200 moles per protein, and is allowed to react at a temperature of 0 to 37xc2x0 C. for 1 hour to 3 days.
Examples of the method for reacting the hydroxyl group in a polypeptide molecule with a polyethylene glycol-polypropylene glycol copolymer derivative include the methods described in Japanese Published Unexamined Patent Application No. 59629/84, Japanese Published Unexamined Patent Application No. 176586/85, WO 89/06546, EP 0539167A2, and the like, and modified methods thereof. That is, specific examples of the methods which can be used include methods wherein a chemical modifying agent selected from a group consisting of polyethylene glycol-polypropylene glycol copolymer derivatives is added to an aqueous solution of a protein which has been adjusted to a pH of 6 to 10 in an amount of 1 to 200 moles per protein, and is allowed to react at a temperature of 0 to 37xc2x0 C. for 1 hour to 3 days.
Modification of at least one hydroxyl group in the polypeptide molecule with a polyalkylene glycol derivative by the above method can provide
(1) a method for the analysis of influences of the hydroxyl group upon the activity of a polypeptide, in a case where the hydroxyl group concerned is in the active site of the polypeptide,
(2) a novel chemical modification method which can avoid diminishing the biological activity of a polypeptide when the polypeptide is treated by a conventional chemical modification method, and a chemically modified polypeptide obtained by the method, and
(3) a novel method which can improve a polypeptide""s resistance to protease, freezing-thawing or denaturing agents, and a chemically modified polypeptide having improved resistance against protease, freezing-thawing or denaturing agents.
The chemically modified polypeptide of the present invention specifically described is an example in which a polypeptide having an hG-CSF activity is used as a polypeptide.
A chemically modified polypeptide in which at least one hydroxyl group in hG-CSF or a hG-CSF derivative is chemically modified with a chemical modifying agent represented by the following formula (Ia) or (Ib) can be exemplified as the chemically modified polypeptide of the present invention.
Chemical modifying agent (Ia):
R1xe2x80x94(OCH2CH2)nxe2x80x94Xxe2x80x94R2axe2x80x83xe2x80x83(Ia) 
{wherein R1, n and X have the same meanings as defined above; and R2a represents 
[wherein R represents OH, halogen or
xe2x80x94Xbxe2x80x94(CH2CH2O)nbxe2x80x94R1b 
(wherein Xb, R1b and nb each has the same meanings as the above X, R1 and n, respectively)]}.
Chemical modifying agent (Ib): 
(wherein R1, M, R3, Z, n and p have the same meanings as defined above; and Wa represents a carboxyl group or a reactive derivative thereof).
At least one molecule of polyethylene glycol derivatives, polypropylene glycol derivatives or polyethylene glycol-polypropylene glycol copolymer derivatives is bonded to a chemically modified hG-CSF or a chemically modified hG-CSF derivative. Thus, the chemically modified hG-CSF or the chemically modified hG-CSF derivative can be used as a mixture or by separating a compound to which one or more molecules are attached.
Separation of the chemically modified hG-CSF or the chemically modified hG-CSF derivative can be carried out using various types of chromatography, such as ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydrophobic chromatography, and the like, and methods, such as ammonium sulfate fractionation and the like, which are generally used for the separation of long chain polypeptides and the like.
The degree of chemical modification can be confirmed by monitoring changes in the mobility of the chemically modified hG-CSF using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method.
The polypeptide content according to the present invention can be measured by the following assay methods.
Assay method 1:
The polypeptide content is measured by the Lowry method [Lowry O. H. et al., J. Biol. Chem., 193:265 (1951)].
Assay method 2:
The polypeptide content is calculated by carrying out SDS-PAGE in accordance with the method of Laemmli [U. K. Laemmli, Nature, 227:680 (1970)], staining the polypeptide separated on the gel with Coomassie Brilliant Blue and then measuring it by a chromatoscanner (CS-930, Shimadzu Corp.).
The chemically modified polypeptide of the present invention can be used as such or in various dosage formulations.
The pharmaceutical preparations of the present invention can be produced by mixing an effective amount of a chemically modified polypeptide as the active ingredient uniformly with a pharmaceutically acceptable carrier. It is preferred that these pharmaceutical preparations are in a unit dosage form suitable for injection administration.
Injections can be prepared as solutions using a chemically modified polypeptide and a carrier comprising distilled water, a salt solution, a glucose solution or a mixture of a salt solution and a glucose solution. They are also prepared as solutions, suspensions or dispersions in the conventional way using appropriate auxiliaries. They can be also prepared as freeze-dried preparations by freeze-drying the solutions. Although freeze-drying conditions are not particularly limited, a freeze-dried product is generally obtained by freezing at xe2x88x9250xc2x0 C. or less for 1 to 5 hours, drying at a shelf temperature of xe2x88x9220xc2x0 C. to 0xc2x0 C. for 24 to 48 hours under a vacuum degree of 50 to 150 mTorr and then drying at a shelf temperature of 10xc2x0 C. to 30xc2x0 C. for 16 to 24 hours under a vacuum degree of 50 to 100 mTorr.
Also, the chemically modified polypeptide preparations can contain various generally used pharmaceutical carriers, fillers, diluents, stabilizers, adsorption preventing agents and the like.
In the case of a chemically modified polypeptide of the present invention, for example a neutrophil and thrombocyte growth enhancing preparation containing a chemically modified hG-CSF or a chemically modified hG-CSF derivative, its dose and its administration schedule are decided depending on its mode of administration, age and body weight of each patient, the disease to be treated and morbid state of each patient; however, a pharmaceutical preparation containing a chemically modified hG-CSF or a chemically modified hG-CSF derivative in an amount of 15 xcexcg to 1.5 mg, preferably 25 to 500 xcexcg, per adult is usually administered 1 to 7 times per week.
Examples of the administration method of the chemically modified polypeptide preparation of the present invention include intravenous injection, subcutaneous injection, and the like, as well as administration as suppositories or nasal drops.
Next, pharmacological activities of the chemically modified polypeptide of the present invention are described by Test Examples.
Growth promoting activity of chemically modified hG-CSF and chemically modified hG-CSF derivatives upon mouse leukemia cell NFS60:
The activity of the G-CSF derivative, chemically modified hG-CSF derivative and chemically modified G-CSF obtained in Reference Example 1 and Examples 5, 14, 17 and 20, which will be described later, to enhance growth of mouse leukemia cell NFS60 [K. Holmes et al., Proc. Natl. Acad. Sci. USA, 82:6687 (1985)] was measured in accordance with the method of Asano et al. [Yakuri to Chiryo, 19:2767 (1991)]. Each compound in respective concentrations shown in Tables 2-1 and 2-2 was allowed to act upon the cells, with the results also shown in Tables 2-1 and 2-2.
Effect to promote recovery of thrombopenia in total body radiation mice:
Four animals per group of male BALB/c mice (6 weeks of age) were radiated to the total bodies (hereinafter referred to as xe2x80x9cRxxe2x80x9d) with 3 Gy per mouse by a 137 Cs radiation source (RI-433, manufactured by Toshiba) and then reared in a clean rack of a specified pathogen-free (SPF) rearing environment facility. They were freely provided with drinking water and feed. As a non-treated control group, mice with no radiation were reared in a manner similar thereto.
Each of chemically modified hG-CSF derivatives obtained in Example 4 which will be described later was dissolved in physiological saline, and the chemically modified hG-CSF derivative solution was subcutaneously administered to mice once on the next day of Rx in a dose of 5 xcexcg/0.2 ml per animal.
Blood samples were periodically collected from the murine vein of eyeground to measure the number of platelets by an automatic blood cell counter (CC-180A, manufactured by Toa Iyo Denshi) The results are shown in Table 3.
In the mice treated with total body radiation of 3 Gy, a considerable decrease in the number of platelets was observed, and the number of platelets became the lowest on 8 to 9 days after Rx and then gradually increased but did not recover to the level before the radiation treatment. On the other hand, decrease in the number of platelets was inhibited in the mice to which the chemically modified hG-CSF derivative was administered, and the number of platelets increased markedly on and after the 8th or 9th day and completely recovered on the 11th or 12th day to the same level before the radiation treatment. The similar effect was observed in a group in which the administration was carried out on the next day and 5th day after Rx.
Leukocyte increasing action in mice:
Using SPF/VAF mice (BALB/cAnNCrj line, males, 8 weeks of age, 4 animals per group) which were preliminarily reared after purchase from Charles River Japan, leukocyte increasing activity in normal mice was confirmed.
Di-substituted obtained in Example 5 (0.34 mg/ml) was diluted to 100 or 10 xcexcg/ml using physiological saline, and subcutaneously administered once at a rate of 10 xcexcl/g (mouse weight). Thus, the dose was 1 or 0.1 mg/kg. As a control group, physiological saline was subcutaneously administered once. Blood samples were collected before the administration and periodically from the next day after the administration, and the number of peripheral blood cells was measured using an automatic blood cell counter (Sysmex F800).
As the results, the number of leukocytes in both administered groups increased to 2.4 to 2.6 times higher level than that of the control group 2 days after the administration. Thereafter, this drug effect attenuated in the 0.1 mg/kg administration group and returned to a similar level of the control group 4 days after the administration, but the number of leukocytes in the 1 mg/kg administration group continued to increase even after 2 days after administration and reached a level 3.5 times higher than that in the control group 4 days after administration. Thereafter, the number returned to a level similar to the control group on the 7th day after the administration.
Examples and Reference Examples are shown below.